Effects Of RNAi Modiated Down Regulation Of S100A4 On Proliferation And Migration Of Pancreatic Cell

Pancreatic cancer is a highly malignant tumor with poor prognosis. Since no specific earlysymptoms exist, most pancreatic cancer has been medium or advanced stage at diagnosis, with5-year survival rate of only 1% to 5%. Incidence of pancreatic cancer is increasing in recent years.Due to poor therapeutic effect of advanced pancreatic cancer, early diagnosis may allow morepatients the opportunity of surgical resection and improved survival. However, current imagingtechnology is still not able to accomplish early diagnosis, and pancreatic cancer-related serologicalmarkers are still not able to accurately reflect tumor status. With the rapid development ofmolecμlar biology, especially the application of DNA recombination technology, many newoncogenes and anti-oncogenes related to pancreatic cancer have been discovered and cloned,providing many new targets for pathogenesis, diagnosis, and treatment of pancreatic cancer.Therefore, we focused on the highly expressed S100A4 gene in pancreatic cancer and RNAitechnology. The effects of S100A4 RNAi on proliferation, apoptosis, metastasis, and invasion ofpancreatic cancer cell line BxPc-3 were systemically investigated, with the aim of providingtheoretical basis for early diagnosis and prognosis evaluation of pancreatic cancer.RNA interference (RNAi) is an in vivo sequence-specific post-transcriptional gene silencingprocess mediated by homologous double-stranded RNA (dsRNA). Since RNAi is a siRNAmediated post-transcriptional gene silencing process targeting exon sequences with no impact on itspromoter or intron sequences, so it is characterized by high specificity, high efficiency and highstability. Now RNAi has been widely used to investigate carcinogenesis and tumor development,invasion and metastasis, cell cycle regμlation, signal transduction, anti-apoptosis gene, andsensitivity to chemotherapy or radiotherapy.S100 proteins are a class of low molecμlar weight (9 ~ 13×103) acidic proteins capable ofdissolving in 100% saturated ammonium sμlfate. They are the largest subfamily of Ca2+ bindingproteins. There are two EF-hands in such proteins with short end sequences. EF-hand is formed byCa2+ selective and highly affinitive helix - loop - helix structure, which is composed of hydrophobichelical structures and hinge region. S100 proteins are usually non-covalent homodimers, which arealso their functional forms. S100A4 (S100 calcium binding protein A4), also known as 18A2, mts1(metastasin), CAPL (calcium protein placental homolog), PEL-98, 42A and p9Ka, is one of the most characteristic member of S100 protein family. S100A4 exists inside the cell as non-covalentdimmer, and secretes outside the cell as covalent dimmer. It has been reported that S100A4 canpromote tumor angiogenesis and therefore plays important roles in carcinogenesis and metastasis.High level expression of S100A4 has been found in a number of tumor cells, which is closelyrelated to metastasis of breast, prostate and colon cancer.S100A4, composed of 101 amino acids and being a member of Ca2+-binding proteinsuperfamily, plays important roles in carcinogenesis and tumor development through Ca2+-binding.High level expression of S100A4 has been found in a number of tumor cells, which is closelyrelated to growth, invasion, and metastasis of tumors. S100A4 is a protein closely related to cellcycle, proliferation, apoptosis, invasion and metastasis.In this paper, we conducted the following researches:1．DesiDesign of S100A4 interference fragments and verification of interference effectsExpression level of S100A4 gene in BxPc-3 cell line was investigated through RT-PCR. Highlevel expression of S100A4 gene was determined in BxPc-3 cell line.Full sequence of human S100A4 gene was discovered in Genebank. 3 pairs of S100A4-siRNAinterfering sequences and negative control sequence were designed and synthesized based onprinciples of siRNA design. S100A4 siRNA 174 was found to show best interfering effects throughWestern Blot verification.Western Blot and Real-Time PCR showed that expression level of target protein and targetmRNA was significantly lowered in treatment group compared with control group, indicatingobvious interfering effects. The results also suggest that RNA interference is specific and efficient.2．Effects of RNAi mediated down regulation of S100A4 on proliferation and apoptosis ofpancreatic cellColony forming rate is the"golden standard"for assessing cell status and proliferationpotential. Colony forming rate was applied in this study to evaluate proliferation status and flowcytometry was applied to investigate apoptotic status of cells. 3 groups were established: untreatedgroup, NC siRNA group, and S100A4 siRNA group. Colony forming rate of S100A4 siRNA groupwas found to be significantly lowered (P<0.05), suggesting inhibition of cell proliferation afterS100A4 gene silencing.Apoptotic status at 24h, 48h, and 72h was compared between 3 groups by flow cytometry. Nostatistical significance was found between these groups, suggesting no effect of S100A4 inhibitionon apoptosis of BxPc-3 cell.3．Effects of RNAi mediated down regulation of S100A4 on migration and invasion of pancreatipancreatic cellTranswell chamber model was employed in this study to investigate migration and invasionability. Transwell chamber comprises upper and lower chambers separated by a permeablemembrane. Cell migration is driven by concentration difference between 2 chambers, so migrationand invasion ability can be compared between cell groups. 3 groups were established: untreatedgroup, NC siRNA group, and S100A4 siRNA group.The amount of cells penetrating membrane was significantly decreased in S100A4 siRNAgroup compared with untreated group and NC siRNA group (P<0.05), suggesting inhibition ofmigration and invasion ability after S100A4 siRNA transfection of BxPc-3 cell.Innovation points of this study: Calcium binding protein S100A4 was targeted, which showedhigh level expression in tumor tissues and low level expression in normal tissues; its effects onproliferation, apoptosis, migration and invasion ability of pancreatic cancer cell BxPc-2 wasthroughly explored by means of RNAi technique; this investigation provides theoretical basis forearly diagnosis and prognosis evaluation of pancreatic cancer, as well as new effective target forcancer therapy; this investigation is beneficial to patients.